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An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA-cathepsin) and to CNBr-activated Sepharose 4B. EPA-cathepsin was active toward the synthetic hexapeptides (Gly-Phe-Leu)2 and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60 degrees C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65 degrees. The specific activity of the preparations (pH 7.0, 65 degrees) was 60-110 percent that of the free enzyme in acid media. Proteolytic activity of the Sepharose-coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr-activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed increased stability.

Peptidase activity of Yersinia pestis cell extract was studied by the hydrolysis of synthetic and some natural substrates, as well as the hydrolysis of fluorogenic aminopeptidase substrates. All peptides and fluorogenic substrates under test were split by Y. pestis cell extract, the splitting of alanylaminopeptidase substrate being linked with at least two enzymes of this cell extract.

Morphofunctional alterations occurring in the colon wall indicate complex interactions of the morphological structures participating in the regulation of the intestinal function in salmonellosis. In the early period (24 hours) of maximum decrease in the mediator activity in the nerve structures the number of enterochromaffin cells and the granulation index increased, while in the period of maximum tension of nerve elements (7 days) a decrease in the enterochromaffin cells and granulation index was noted. At later periods (21 days) a relative morphofunctional stabilization of the mucous components and the intramural ganglia has been revealed.

Fractionation of proteins of aqueous and salt (1 M KCl) extracts of human spleen on Sephadex G-100 revealed several proteinases hydrolyzing histone and casein and active in a neutral medium. The enzymes were found in the extracts in a relatively inactive form, due to the presence of an inhibitor, chiefly in the aqueous extract. Proteinases active in a neutral medium were found in two protein fractions. “High-molecular-weight” proteinases are inhibited by di-isopropylfluorophosphate (DFP), so that they can be classed in the group of serine proteinases. The fraction of “low-molecular-weight” proteinases contains neutral SH-dependent proteinase (proteinases) and enzymes inhibited by DFP. Kininogenase activity and activity hydrolyzing N-Boc-L-alanine nitrophenyl ester, N-benzoyl-L-tyrosine ethyl ester, and N-benzyl-DL-arginine-p-nitroanilide also were found in this fraction.

Administration of monoamine oxidase type A inhibitor clorgyline to rats before hyperoxia prevented oxygen-induced increase in diene conjugate and Shiff's base brain and plasma levels in hyperoxia. This was due to antioxidative effect of clorgyline which resulted in stabilization of blood cellular membranes. Clorgyline had a normalizing effect on extraerythrocyte hemoglobin level, total peroxidase activity and glucose-6-phosphate dehydrogenase activity in the serum.

The inhibitory effect of kininogen (especially its high molecular form) on kallikrein esterase activity and on the process of kallikreinogen auto-activation is demonstrated. Kallikreinogen and kininogen from human blood serum partially purified on QAE-Sephadex. CM-Sephadex and G-200 Sephadex were used in experiments.

A chromatographic method is developed for quantitative estimation of the collagenase-like enzyme (CLE) activity in extract of adenohypophysis and in preparations obtained during various steps of the enzyme isolation. The enzymatic hydrolysis of Cbz-Gly-Pro-Ala-Gly-Pro-Gly-OCH3 in presence of 2-mercaptoethanol at pH 8.0 was used as a pattern. The products formed were separated by chromatography on the paper; then they were stained with ninhydrin and converted into cupric complexes during extraction with ethanol; the optic density was measured at 510 nm. The optimal conditions for the enzymatic reaction were established. The method enabled to estimate the CLE activity in presence of prolyl carboxypeptidase. The specific effect of the CLE purified preparation on various synthetic peptides is discussed.

Properties of the carboxycathepsin inhibitors teprotide, captopryl, enalacryl are considered. Presence of low molecular thermo- and acid stable carboxycathepsin inhibitors of peptide nature in blood serum and lymphocytes is discussed.

Using the fluorimetric method, the activity of dipeptidyl-carboxypeptidase (DCP) was studied in 85 patients suffering from essential hypertension. It was ascertained that the DCP activity in the blood serum of such patients was significantly higher than in normal subjects. The highest activity of the enzyme was observed in cases where essential hypertension was aggravated by chronic heart failure or an acute impairment of the cerebral circulation. Inflammatory processes in hypertensive patients were associated with a considerable fall in the serum enzyme activity.

The kininogenase activity of highly purified preparations of cathepsins D from human liver and spleen, leukemic infiltrate obtained from patients with myeloic leukemia, and from chicken liver was studied. It was found that pepstatin, a specific inhibitor of carboxylic proteinases, inhibits this activity of cathepsins D. Interaction of chicken liver cathepsin D with human plasma substrate, which is possibly a low molecular weight kininogen (Ks = 1.3.10(-7) M) results in a production of the bradikinin analog methionyl-lysyl-bradikinin. The role of cathepsins D as potent inflammatory agents responsible for the generation of biologically active peptides--mediators of inflammation from the protein substrates including kininogens under desintegration of lysosomes is discussed.

Two SH-dependent proteinases (I and II) active in neutral media were isolated from bovine spleen and purified to apparent homogeneity. The histone-hydrolyzing activity of proteinase I was increased 3500-fold as compared to that of the original extract. Proteinase I hydrolyzed a variety of proteins (histones, azocasein, hemoglobin, collagen) but did not hydrolyze low molecular weight synthetic substrates, such as BAPA, BANA, BAEE, ATEE, Leu-beta-NA, Arg-beta-Na and Ala-beta-NA. The molecular weight of the enzyme as determined by SDS electrophoresis was found to be about 23,000. Isoelectrofocusing of the enzyme resulted in one major component with pI of 6.05 and in two minor components with pI of 6.2 and 6.4. Proteinase II hydrolyzed Leu-beta-NA, Arg-beta-NA and Ala-beta-NA but did not hydrolyze beta-naphthylamides of dicarboxylic acids and Gly-Phe-beta-Na. This proteinase split BANA and histone and very slowly split azocasein and collagen. Proteinase II was found to have a molecular weight of 30 000 and a pI of 6.8-6.9. Proteinase I inactivated fructose-1.6-diphosphate aldolase, partly inactivated glucose-6-phosphatase dehydrogenase and caused activation of phosphodiesterase of cyclic nucleotides. Proteinase II had no effect on the activity of the above enzymes. A comparison of proteinase I and II with enzymes described in literature demonstrated that the former was cathepsin L, while the latter was cathepsin H from spleen.

The paper deals with the isolation of cathepsin D from rat liver, chicken liver and bovine spleen by affinity chromatography. The synthesis of the adsorbent was performed using the competitive inhibitor of cathepsins D and pepsin pepstatin and activated Sepharose. Application of a pepstatin-Sepharose column allows obtaining a highly active and purified enzyme in a short period of time.

2200-fold purified homogenous preparation of carboxycathepsin (peptidyl-dipeptidase) is isolated from bovine lung. The enzyme isolated converts angiotensine I into angiotensine II and distroys bradikinin. It is active in neutral medium, is activated by chloride ion and is inhibited by EDTA and Middle Asian snakes venom. The molecular weight of the enzyme is 180 000-190 000 as estimated by means of polyacrylamide gel electrophoresis, its isoelectric point is 4.48-4.53. The comparison of properties and specificity of carboxycathepsin from bovine lung and kidney draws to the conclusion that both enzymes are identical.

A content of neutral sugars and N-acetyl-glucosamine in homogeneous cathepsin D preparations from a variety of vertebrate organs was determined. A more detailed study of the carbohydrate component was carried out with chicken liver cathepsin D preparation. It was shown that carbohydrates constitute 20% of the molecule of this cathepsin and contain glucosamine (11.6%) and mannose (10%). Removal of the major portion of the carbohydrates by treatment with mixture of glycosidases did not lead to any significant decrease of biological activity. This finding suggests that the carbohydrate component is not essential for the biological activity of the enzyme. Analysis of distribution of carbohydrates in the peptides of the trypsin hydrolyzate of cathepsin D allows conclusion that the enzyme molecule has several carbohydrate chains attached to different sites of the molecule.

The interaction of clostridiopeptidase of Clostridium histolyticum with EDC, TNM and MA, the specific reagents for COOH-groups, tyrosine and lysine residues was studied. It was shown that at pH 6.0 EDC inactivates the enzyme. The inactivation process follows the pseudo-first order kinetics and is described by a second order rate constant equal to 1 M-1 min-1. The synthetic substrate does not prevent, in practical terms, the enzyme inactivation by EDC. At pH 8.0 TNM modifies about 19 tyrosine residues in the clostridiopeptidase molecule which is accompanied by marked inhibition of the enzyme activity (down to 70-90%). In this case, the inactivation process is not described by simple pseudo-first order kinetics but is characterized by two steps (fast and slow) with second order rate constants of approximately 14 and 3.5 M-1 min-1, respectively. The synthetic substrate partly prevents the inactivation of the enzyme by TNM and protects 11 tyrosine residues. The MA-induced incorporation of 13 +/- 3 maleyl groups into the clostridiopeptidase molecule in partially prevented by the synthetic substrate with protects the enzyme against inactivation. The data obtained suggest that lysine residues are seemingly included into the active center of clostridiopeptidase, whereas tyrosine residues provide for the maintenance of active conformation of the enzyme.

Aminopeptidase A, which specifically hydrolyses N-terminal dicarbonic amino acid residues containing free alpha-amino groups, is isolated from bovine kidney. The enzyme is 500-fold purified and is homogenous under electrophoresis and ultracentrifugation. Aminopeptidase A has pH optimum of 7.5, it is activated with Ca2+ and inactivated with EDTA. Its molecular weight is 53000. The enzyme hydrolyses alpha-L-aspartyl-beta-naphtylamide and splits peptides having N-terminal glycine, lysine, arginine and alanine are hydrolyzed by the enzyme much slower. Aminopeptidase A does not attack alpha-L-alanyl-beta-naphtylamide, leucineamide, insulin, peptides with blocked N-terminal amino acid and peptides which have proline to be the second N-terminal amino acid.

Four highly purified enzyme preparations, which belongs to the cathepsine A group in their substrate specificity, are isolated from the extract of a hen liver acetone powder. The preparations are designated as A1, A2, A3 and A4 according to their electrophoretic mobility. The A1 component is a protein with molecular weight of about 200 000, it degrades a synthetic substrate and, to a small degree, hemoglobin. This protein is suggested to be the fragment of a liver enzyme complex. The A2 component has molecular weight of about 70 000 and the highest activity. The A3 component has molecular weight 100 000 and the lowest activity. The A2 and A3 components are similar to cathepsine A from other tissues. THe A4 component is characterized by a high electrophoretic mobility, a resistance in neutral and weakly alkaline media and a low molecular weight (of about 60 000). Cu2+, Ag+ and diisopropylphosphofluoridate completely inhibited the activity of all the enzyme preparation studied. Tosyl-alpha-amino-phenylethyl-chloremethylketone inhibited the enzymes activity only by 50-70%.

The collagenases (I, II and III) have been obtained in a highly purified state from fresh cultural medium of Clostridium histolyticum. The collagenases were similar in their properties to clostridiopeptidase A. The three enzymes differed in their molecular weights, isoelectric points and in some chemical properties. Collagenase II exhibited the most potent hydrolytic activity. Its collagenolytic activity was two-fold higher and the peptidase activity was twenty-fold higher as compared with that of collagenase I. All the three enzymes were inactive towards azocasein and were inhibited by EDTA and cysteine.

The action of two earlier isolated highly purified spleen thiol proteinases on angiotensins I and II, bradykinin and kallidin was investigated. It was demonstrated that proteinase I which is apparently cathepsin L from bovine spleen brings about rapid inactivation of angiotensin II with a splitting of the Tyr-Ile bond and a formation of two tetrapeptides. Proteinases I also split angiotensin I. Proteinase I partially inactivates bradykinin and kallidin by splitting the Gly4-Phe5 bond. The activity of proteinase I toward angiotensin II is about 50 times higher than that toward bradykinin. The corresponding values of Km and V are 7.5 X 10(-5) M and 10.0 mumole/min/mg. The possible role of proteinase I in angiotensin II inactivation under physiological conditions is discussed. Proteinase II converts kallidin to bradykinin by splitting off the N-terminal lysine. Proteinase II causes partial inactivation of bradykinin by splitting of the Gly4-Phe5 and Phe5-Ser6 bonds of this peptide. Proteinase II possesses both aminopeptidase and endopeptidase activities and is therefore cathepsin H from spleen. Proteinase II does not split either angiotensin I or angiotensin II.

A method for isolation of glutamin (asparagin) ase from Pseudomonas aurantiaca BKMB-548 has been developed. The enzyme preparation is homogeneous during polyacrylamide gel electrophoresis (pH 7.5 and 7.0) with SDS. The pH-optima of the enzyme thermal stability and of the glutaminase activity are equal to 6.0-8.0. At higher pH values the asparaginase activity increases within the pH range of 4-9. The amino acid composition of glutamin(asparagin)ase has been determined.

4-methoxy-beta-naphthylamide and 7-amino-4-methyl coumarin, derivatives of Z-Ala-Arg-Arg, Leu- and Gly-Phe-beta-naphthylamides were used as substrates in estimation of peptide hydrolases activity in blood serum of patients with malignant tumors and glomerulonephritis in order to ascertain their efficiency for diagnostic purposes in clinic. Each of the fluorogenic substrates studied was hydrolyzed by various peptide hydrolases from blood serum both under normal and pathological conditions: metallopeptidases, cysteine- and serine-dependent peptide hydrolases. The rate of Z-Ala-Arg-Arg-MNA hydrolysis was decreased in lung, kidney and ileum cancer as well as in glomerulonephritis as compared with normal state. The "alkaline-resistant" cysteine-dependent cathepsin B-like proteinase, hydrolyzing this peptide, was not detected in blood serum neither in normal state nor in these diseases studied. Leu-NA and Gly-Phe-NA were hydrolyzed most effectively in blood serum of patients with lung cancer and glomerulonephritis as compared with normal state; cysteine-dependent peptide hydrolases were most markedly activated. Alterations in the enzymatic activity, detected in blood serum, did not exhibit any specificity for definite diseases, they were observed both in malignant and inflammatory impairments. The data obtained suggest that the fluorogenic substrates studied could not be suitable for clinico-diagnostic purposes.

To investigate conformations of peptide inhibitors of the angiotensin-converting enzyme in the enzyme-inhibitor complex, the synthesis, studies of inhibitory activity, and conformational calculations of analogues of bradykinin-potentiating peptides with N-methylalanine or D-alanine in place of L-proline or L-alanine residues have been carried out. All the analogues showed a sharp decrease of inhibitory activity in comparison with the natural peptides, that might be considered as an indirect confirmation of the earlier proposed "conformation of inhibition" of the above-mentioned peptides.

Apart from the generally known functions, the heart has also an endocrine function. Atrial cardiocytes, being typical secretory cells, release peptide hormones into the blood stream: atrial natriuretic peptide containing 28 amino acids and cardiodilatin. The structure of atrial peptides was determined. It was shown that both peptides were derived from their common precursor, a protein containing 151 amino acids. The presence of specific receptors is demonstrated on plasmatic membranes of cells of kidney epithelium, arterial smooth muscle, arterial endothelium, kidney cortex and hypophysis. The interaction of atrial peptides with these receptors activates the guanylate cyclase system. The biological action of atrial peptides manifests itself in the quick, massive and instantaneous increase of diuresis and electrolyte excretion, elevated clearance of creatinine, decrease of kidney vascular resistance, intensification of glomerular filtration, inhibition of stimulated secretion of aldosterone, relaxation of blood vessels, elimination of arterial and intestinal spasm induced by various endogenous and exogenous vasoconstrictors and in correction of kidney hypertension. Various radioimmunoassays for the presence of atrial peptides in human plasma were developed; it was shown that in patients with congestive heart failure the content of atrial peptides is increased.

Cathepsins D were isolated from human liver and spleen, from three malignant tumours (kidney cancer, sarcoma, spleen tumour caused by chronic myeloleucosis) and from one non-malignant tumour (uterine myoma). The isolation procedure involved adsorption on pepstatin-Sepharose and gel-filtration on Sephadex G-100. A comparative study of the properties of these enzymes was carried out. The molecular weights, specific proteolytic activity toward hemoglobin and the synthetic hexapeptide Acetyl-Val-Val-Leu-Ser-Leu-Thr, carbohydrate content and type of dissociation of the molecules into polypeptide fragments during electrophoresis in the presence of DS-Na were determined. All the enzymes under study had molecular weights of about 45 000 except uterine myoma cathepsins (mol. weight 95 000). The latter cathepsin also differed from the other enzymes in its higher carbohydrate content.

Carboxycathepsin from bovine kidney split the dipeptide fragments from the C-terminal part of peptides of different structure. Peptides containing the proline residue at the second position from the C-terminal amino acid residue and also peptides with substituted terminal alpha-carboxyl group were not hydrolyzed by carboxycathepsin. The enzyme was activated by Cl, Zn2+, Co2+ and Mn2+. The substances which formed the chelate complexes with ions of two-valent metals and also heavy metal ions, inhibited the enzymatic activity. Diisopropyl fluorophosphate did not inhibit carboxycathepsin. The homogeneous preparation of carboxycathepsin converted angiotensin 1 into angiotensin 11 and hydrolyzed bradikinine, splitting off C-terminal dipeptides consequentially.

The experiments were carried out to elucidate the effect of carboxycathepsin (CC) activity inhibition by a specific inhibitor--captopril--on plasma enzyme concentration in normotensive rats and rats with renovascular hypertension. A single oral administration of captopril (10 mg/kg body weight) produced an increase in CC concentration in both hypertensive and sodium-depleted normotensive rats with a parallel decrease in arterial pressure, but had no effect on sodium-repleted normotensive rats. It is suggested that the increase in plasma CC concentration is a compensatory response to the inhibition of CC activity by captopril; it is also possible that the increase observed reflects the state of renin-angiotensin system.